Towards a characterization of the structural determinants of specificity in the macrocyclizing thioesterase for deoxyerythronolide B biosynthesis.

Type I polyketide synthases (PKSs) are giant multidomain proteins that synthesize many therapeutics and other natural products. The synthesis proceeds by a thiotemplate mechanism whereby intermediates are covalently attached to the PKS. The release of the final polyketide is catalyzed by the terminal thioesterase (TE) domain through hydrolysis, transesterification, or macrocyclization. The PKS 6-deoxyerythronolide B synthase (DEBS) produces the 14-membered macrolide core of the clinically important antibiotic erythromycin. The TE domain of DEBS (DEBS TE) has well-established, empirically-defined specificities for hydrolysis or macrocyclization of native and modified substrates. We present efforts towards understanding the structural basis for the specificity of the thioesterase reaction in DEBS TE using a set of novel diphenyl alkylphosphonates, which mimic substrates that are specifically cyclized or hydrolyzed by DEBS TE. We have determined structures of a new construct of DEBS TE alone at 1.7Å, and DEBS TE bound with a simple allylphosphonate at 2.1Å resolution. Other, more complex diphenyl alkylphosphonates inhibit DEBS TE, but we were unable to visualize these faithful cyclization analogs in complex with DEBS TE. This work represents a first step towards using DEBS TE complexed with sophisticated substrate analogs to decipher the specificity determinants in this important reaction.

Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13.

Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction.

Crystallization and preliminary crystallographic studies of both components of the staphylococcal LukE-LukD leukotoxin.

Soluble forms of recombinant LukE protein (expressed in Escherichia coli) and of wild-type LukD protein (expressed in Staphylococcus aureus), which together form the staphylococcal LukE-LukD leukotoxin, were purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The crystals of LukE belonged to space group I4, with unit-cell parameters a = b = 134.50, c = 64.43 Å, and diffracted X-rays to 1.6 Å resolution. The crystals of LukD belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 48.04, b = 50.99, c = 137.40 Å, and diffracted to 1.9 Å resolution. Molecular replacement using the LukF-PV structure (PDB entry 1pvl) as a template model allowed the identification of an initial structure solution for the LukD data. In the case of LukE, a solution comprising only a single copy of the search model (LukS-PV; PDB entry 1t5r) was found, although the unit-cell parameters indicated that up to three molecules could be accommodated in the asymmetric unit.